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Background: Several studies have demonstrated that the pathophysiological and morphological changes in early diabetic nephropathy were mediated by an increase or decrease in nitric oxide (NO) production and/or activity. There are few reports suggesting a relationship between NO and the renin–angiotensin system.

Aim of the work: The present study was designed to determine the effects of early diabetic state on NO production and also to assess the possible effects of angiotensin receptor blockers (ARBs) on these changes.

Materials and methods: Thirty adult male albino rats were included in this study. Twenty were injected with streptozotocin for induction of diabetes. The other 10 were injected with the vehicle and served as control. Two days after injection, the diabetic animals were randomly divided into two groups of 10 animals each. One group was given valsartan as an ARB and the other group received no further treatment. Three weeks later, all animals were sacrificed and the kidneys were processed for obtaining paraffin sections. The sections were stained with H&E, Masson’s trichrome, and periodic acid–Schiff. The sections were also stained with an immunohistochemical stain against endothelium-derived nitric oxide synthase (eNOS).

Results: Diabetes induced histological changes in the form of glomerular hypertrophy, increased glomerular matrix, focal areas of tubular atrophy, medullary congestion, and slight fibrosis. Immunostaining was present in the control kidney in the glomeruli and in the collecting tubules of the medulla. Diabetes induced a positive reaction in the proximal and distal convoluted tubules and increased immunoreactivity in the collecting tubules. Treatment with valsartan resulted in an improvement in the morphology of the kidney and a reduction in the intensity of eNOS immunostaining.

Conclusion: NO increases in early diabetic kidney and ARB in the form of valsartan could be recommended for preventing the development of diabetic nephropathy.

Introduction

Diabetic nephropathy (DN) is one of the serious complications of diabetes and a common cause of end-stage renal failure worldwide. In recent times, the number of patients requiring dialysis for DN has increased. Hence, nephropathy, which is a severe and chronic disorder, influences the quality of life and prognosis of diabetic patients 1,2. Preventing the occurrence and slowing down the development of DN has therefore become a very important issue because early stages of DN are accompanied by glomerular hypertrophy and hyperfiltration leading to overt nephropathy 3. Endothelium-derived nitric oxide (eNO) has been identified as one of the compounds responsible for alterations in glomerular filtration 4. NO is a paracrine mediator with a wide spectrum of physiological actions, including the control of vascular tone, antithrombotic actions, cell cycle regulation, neurotransmission, and inflammation. NO is synthesized during conversion of its physiological precursor l-arginine to l-citrulline. This reaction is catalyzed by an enzyme known as endothelium-derived nitric oxide synthase (eNOS) 5. Meanwhile, evidence has suggested that there is activation of the intrarenal renin–angiotensin system in diabetes mellitus. Furthermore, although renin release has been shown to be modified by the eNO system, the interaction between the two systems in diabetes has not been fully explored 6. Angiotensin II type 1A receptor blockers (ARBs) were shown to ameliorate endothelial-dependent vasodilatation in diabetic patients 7. Thus, this study aimed at investigating changes in eNo during the early stages of experimental diabetes in rat kidney through immunolocalization of the enzyme eNOS; the study also aimed at evaluating the renoprotective effects of ARB and its interaction with eNOS.

Materials and methods

Drugs

Streptozotocin (STZ; Sigma, St Louis, Missouri, USA): this was administered in the form of a single intraperitoneal injection at a dose of 60 mg/kg dissolved in 0.5 ml of citrate buffer (0.1 mol/l) for induction of diabetes mellitus 8.

Valsartan (Tareg Tablets 40 mg; Novartis, SAE, Cairo, Egypt): it is a selective ARB given at a dose of 20 mg/kg/day by gavage. Taking into consideration the fact that valsartan is a well-known antihypertensive drug, the dose used was the least effective dose to minimize the blood pressure-lowering effect on the animals as much as possible 9.

Animals and treatment protocols

The present study included 30 adult male albino rats of 180–230 g body weight; they were obtained from and housed in the animal house of Kasr-El-Aini Faculty of Medicine, Cairo University. This study was approved by the ethics committee for animal research in the animal house of Kasr-El-Aini Faculty of Medicine, Cairo University, Egypt, following international ethics and regulations for animal research in laboratory applications 10.

The animals received a standard diet for rodents and were allowed free access to water. They were distributed into three groups each one containing 10 rats.

Diabetes was induced in 20 animals through a single intraperitoneal injection of STZ. The remaining 10 animals (control) were injected with the same volume of vehicle (group I).

Diabetes was confirmed by measuring their tail blood glucose concentrations 48 h after STZ injection. Rats with blood glucose over 300 mg/dl were considered diabetic 11. The diabetic animals were divided into two equal groups; group II did not receive any further treatment and group III received valsartan once daily.

Evaluation methods

Three weeks after induction of diabetes all animals were anesthetized through a subcutaneous injection of thiopental sodium at a dose of 50 mg/kg and then sacrificed. The right kidneys were dissected out, fixed in 10% formalin solution, embedded in paraffin, and cut at 6 μm thickness. The sections were stained and studied as follows:

Histological study

The sections were stained with H&E to examine the structural changes, with periodic acid–Schiff (PAS) to identify the changes in the mesangial matrix and the basement membranes, and with Masson’s trichrome to demonstrate collagen fibers.

Immunohistochemical study

Immunohistochemical staining was carried out using the primary antiserum to eNOS (Cat. #RB-1711, NeoMarkers; Lab Vision Corporation, Fremont, California, USA). Tissue sections were boiled in 10 mmol/l citrate buffer (pH 6.0) (Cat. #AP-9003, NeoMarkers) for 20 min followed by cooling at room temperature for 20 min and washing in Tris-buffered saline. Thereafter, the sections were incubated overnight with the primary antibody at 4°C. A standard labeled streptavidin–biotin immunoenzymatic antigen detection procedure recommended by the manufacturer was followed using goat antimouse IgG and the sections were then counterstained with Mayer’s hematoxylin. Cells positive for eNOS showed cytoplasmic brown deposits. Positive control tissue was obtained by immunostaining a section of endothelial positive cells. Negative control sections were obtained by omission of incubation with the primary antibody (NeoMarkers) 12.

Morphometric study

Data on the following were obtained using a Leica Qwin 500 image analyzer computer system (Leica, Hessen, Germany):

Glomerular changes:

Glomerular matrix index: the glomerular matrix index represents the ratio of the mesangial matrix area to the glomerular tuft area in PAS-stained sections. It is used to determine the degree of increased glomerular matrix. It was measured as the percentage of the area positive for PAS reaction within the glomerular tuft in 10 randomly selected glomeruli per animal.

Glomerular fibrosis: to evaluate fibrosis in the glomeruli, Masson’s trichrome-stained sections were used. The percentage of area stained blue was measured per unit tuft area in 10 randomly selected glomeruli per animal.

Cortical interstitial fibrosis: cortical interstitium was defined as the peritubular space and included the tubular basement membrane and peritubular capillaries. Interstitial fibrosis was estimated by measuring the percentage of area occupied by the Masson trichrome-positive interstitium in 10 randomly selected nonoverlapping fields per animal.

Medullary fibrosis: it was estimated by measuring the percentage of trichrome-positive area in 10 randomly selected nonoverlapping fields of the renal medulla per animal.

Immunoreactive optical density: the optical density was determined randomly in the cortex and the medulla. Measurements were taken in 10 randomly selected nonoverlapping fields at magnification ×400 from each animal.

Statistical analysis

Statistical analysis was performed on Excel software. Data were presented as mean±SD. Differences among the study groups were detected by one way analysis of variance as the global test to determine any differences in data prior to comparing pairs of groups using t-test to compare each two groups. P values <0.05 were considered statistically significant 13.

Results

Histological results

Control animals showed a normal appearance for the glomeruli composed of capillary tufts surrounded by Bowman’s capsule with space between the visceral layer adherent to the capillary tuft and the parietal layer, as well as proximal convoluted tubules with characteristic narrow lumen lined by a few cuboidal epithelial cells and distal wider tubules lined by more low cuboidal epithelial cells and the medulla containing wider collecting tubules with a wide lumen and thin wall lined by cubical cells. Glomeruli and tubules were crowded in the kidney with minimal interstitial tissue in between (Fig. 1). Diabetic animals demonstrated glomerular enlargement in most of the glomeruli. Some of them were markedly enlarged. Adhesions of the glomerular tuft with some areas of the Bowman capsule were seen. Some glomeruli demonstrated marked mesangial expansion so that Bowman’s spaces were almost completely obliterated. Tubulointerstitial changes were present, in the form of focal tubular dilatation, and vacuolization of the lining cells with appearance of casts inside the tubules. The medullary region demonstrated vascular congestion among the collecting tubules (Fig. 2).

Animals treated with valsartan demonstrated less severe changes. Some glomeruli were enlarged, whereas others appeared of normal size. Bowman’s spaces were still narrow. Congestion was still present in the glomeruli. Most tubules appeared normal. No remarkable capillary congestion was seen in the medulla (Fig. 3).

With respect to PAS staining, the control group showed staining reaction in the mesangial matrix in the glomeruli and boundary of Bowman’s capsules and staining of the basement membrane of proximal, distal, and collecting tubules (Fig. 4). The extracellular matrix of the diabetic group appeared to be increased in the mesangium in the glomeruli with thickened parietal layer of Bowman’s capsule in some areas. The basement membranes of the convoluted and collecting tubules appeared thickened as well (Fig. 5). The group treated with valsartan demonstrated less increase in the staining intensity of the glomerular tuft and apparently normal parietal layer of Bowman’s capsule. The basement membranes of the tubules appeared normal (Fig. 6).

With respect to Masson’s trichrome staining, the control group showed staining reaction in the mesangial matrix in the glomeruli and boundary of Bowman’s capsule and staining of the basement membrane of proximal, distal, and collecting tubules (Fig. 7). In the diabetic group, connective tissue fibers appeared increased in the glomeruli, with thickened parietal layer of Bowman’s capsule in some areas. The basement membranes of the convoluted and collecting tubules appeared thickened as well (Fig. 8). The group treated with valsartan demonstrated a slight increase in the connective tissue content compared with control but the amount was still less than that of the diabetic group (Fig. 9).

Immunohistochemical staining and intensity

The control group demonstrated absent immunoreaction for eNOS in the proximal and distal convoluted tubules. The capillary tufts of most glomeruli were immunoreactive. The collecting tubules in the medullary region showed weak immunoreactivity in most of the collecting tubules (Fig. 10).

The diabetic group demonstrated positive eNOS immunoreaction in the glomerular tufts. There was strong reaction in most tubules occupying the whole cytoplasm in the proximal and distal convoluted and collecting tubules (Fig. 11).

In the group treated with valsartan, most glomeruli were immunoreactive. Both the proximal and distal convoluted tubules showed variable degrees of reaction. Some tubules demonstrated weak reaction in the basal parts of the cytoplasm. Other tubules demonstrated moderate reaction. A few tubules were intensely stained. Nonreactive tubules could also be observed. The collecting tubules of the treated group demonstrated moderate reaction (Fig. 12).

Morphometric results

The diabetic group demonstrated a significant increase in the glomerular matrix index, in glomerular collagen fibers, interstitial fibrosis, and medullary fibrosis, and in the optical density of immunostaining in both the cortex and the medulla when compared with the control group. The treated group demonstrated a significant reduction in these parameters when compared with the diabetic group but they were still significantly higher than those of the control group (Charts 1 and 2 and Tables 1 and 2).

Discussion

DN is one of the major causes of end-stage renal failure. The mechanism of development of abnormal hemodynamics in this disorder is unclear. Clarification of the pathogenesis of DN and the development of novel and effective therapeutic strategies are therefore very important 14. This study aimed at studying the role of NO in early diabetic changes in the kidney and also at evaluating the efficacy of ABR using valsartan in the prevention of these changes.

In the present study, diabetic rats exhibited diffuse glomerular enlargement, mesangial expansion and congestion, obliteration of Bowman’s spaces, increased glomerular matrix, and thickened basement membrane of the parietal layer of Bowman’s capsule. Similar findings were reported in many previous studies. These changes were defined as glomerulosclerosis and may be attenuated by valsartan 15.

Consistent with the present findings, glomerulosclerosis has been reported to be a characteristic of experimental diabetic animals and humans with diabetes mellitus. Recent advances in renal pathophysiology strongly suggest that the diffuse expansion of the mesangial region may play a critical role in the obliteration of the capillary lumen, leading to a reduction in the surface area available for filtration and ultimate cessation of glomerular function in various forms of glomerulopathy including diabetic glomerulosclerosis 2,14. These alterations appear to be homologous to the early features found in the human diabetic kidney with increased NO level in which glomerular hypertrophy with mesangial expansion is claimed to be a central pathology 5.

The present study demonstrated that diabetes induced tubular changes in the form of tubular dilatation, vacuolization of lining cells, tubular casts in addition to vascular congestion, and slight fibrosis. Previous reports demonstrated similar findings 16.

The present study demonstrated eNOS expression both in the glomerular tuft and in the collecting tubules of the medulla of the control kidney. Previous reports demonstrated similar findings in which NO was mainly present in the renal medulla 4, whereas others reported that NO, a free radical in the form of a highly diffusible gas, exerts a wide spectrum of physiological actions, including the control of vascular tone, antithrombotic actions, cell cycle regulation, neurotransmission, signal transduction, and inflammation. There is now abundant evidence that physiological levels of NO have a crucial role in the maintenance of renal hemodynamics, renal perfusion, and glomerular filtration in the normal kidney 17.

In the present study, immunostaining intensity for eNOS was higher in diabetic rats than in control animals in the renal medulla. Moreover, the proximal and distal convoluted tubules developed positive immunoreactivity, which was decreased after valsartan treatment. Similar findings have been demonstrated by other authors 18.

NO produced by eNOS of the kidney could induce hyperfiltration and an increase in the glomerular volume; thus, NO was considered a potential candidate for mediating the early diabetes and vascular permeability 19.

Increased glomerular blood flow cannot necessarily increase intraglomerular pressure and therefore the diabetic kidney abnormally regulates intraglomerular pressure, with imbalance between afferent and efferent arteriolar vasodilatation, leading to increase in glomerular pressure and allowing systemic hypertension to be transmitted to the glomerulus 20.

Increased renal NO production could be explained by disturbance in the amino acid metabolism in diabetes. Elevated glucose induces increased intracellular levels of diacylglycerol, leading to activation of protein kinase C, which has been shown to activate NOS. This pathway may represent the missing link between hyperglycemia and hyperfiltration 21.

Oxidative and nitrosative stresses are involved in a nonenzymatic reaction between sugar and other carbonyl compounds with long-lived matrix proteins, leading to the formation of a group of molecules known as advanced glycosylation end products (AGEs). Each AGE structure has its own formation mechanism and thus its own dependence on oxidative stress 22.

Hyperglycemia, AGE, and diabetes create environments favoring extracellular matrix (ECM) deposition. Increased collagen fiber deposition was also demonstrated in the interstitial tissue of the cortex and around the collecting tubules 23.

Large amounts of NO have also been implicated in the renal abnormal vasodilatation with inflammation. NO produced in large amounts has been suggested to cause direct cytotoxic effects on the endothelium. Another effect of NO may be through DNA damage. The interaction of NO and the superoxide anion generates peroxinitrite, which induces lipid peroxidation and cytotoxicity 24,25.

In contrast to the implication of the present observations of reactive increase in NOS in diabetic rat kidney, many investigators have reported that endothelium-dependent, NO-mediated relaxation of the arteries has decreased in diabetic animals and inferred that NO production is decreased 26,27.

In the present study, we investigated the efficacy of early intervention with ARBs such as valsartan in preventing the development of diabetic renal changes in rats. Treatment was commenced immediately after diabetes induction and animals were followed up for 3 weeks. ARB slowed down mesangial expansion and reduced the development of glomerulosclerosis. Also, tubulointerstitial and medullary affection was minimized.

The present results are consistent with previous studies on diabetic and nondiabetic nephropathies. The studies on diabetic and nondiabetic renal disease have indicated that an initial reduction in proteinuria after the onset of antihypertensive medication predicts the long-term preservation of kidney function and that ARBs may have long-term renoprotective effect 28.

In agreement, ARBs significantly retarded the rate of loss of renal function in a group of patients with DN. This could be because ARBs may interfere with the trophic properties of angiotensin II to promote cellular and glomerular hypertrophy or diminish the accumulation of ECM. Either of these processes could be an important initial step leading to glomerular scarring 29.

The present study demonstrated that treatment with valsartan led to decreased NOS immunoreactivity. This result was consistent with previous studies showing increased expression of eNOS 2 weeks after STZ-induced diabetes and this was attenuated by ARB 11.

Angiotensin II has been reported to upregulate the in-vitro synthesis of NOS in various cell types, including glomerular endothelial cells, mesangial cells, and tubular epithelial cells. Therefore, blockade of angiotensin II receptors by ARB may decrease the generation of NO 30,31.

In summary, this study indicates that the increase in eNOS expression in the kidney possibly participates in the regulatory pathways activated by diabetes mellitus. Intervention studies to block eNOS are needed to address this hypothesis directly. Also, the present results suggest that ARBs represent valuable drugs in the treatment of DN. More investigations are needed to clarify the complex pathophysiology for further therapeutic intervention.